Streptavidin is used ubiquitously in biology because of the affinity and stability of its binding to biotin (Green, N. M., Methods in Enzymol. 184: 51-67, 1990). Streptavidin binds biotin with a femtomolar dissociation constant (Green, N. M. Methods in Enzymol. 184: 51-67, 1990). This tight and specific binding has led to the use of streptavidin for labeling and purification of biotinylated proteins, DNA and cells, for targeting of therapeutics proteins and drugs, for assembly of nanodevices. However, streptavidin is a tetramer, which permits binding to multiple binding sites and can result in cross-linking of the bound molecules. The occurrence of cross-linking makes streptavidin tetramers unsuitable for many applications. Mutations that make the streptavidin protein monomeric do reduce cross-linking, but also reduce the biotin binding affinity by 104 to 105-fold (Qureshi, M. H. et al., J Biol. Chem. 276: 46422-46428, 2001; Green, N. M. and Toms, J. C., Biochem. J. 133: 687-700, 1973; Laitinen, O. H. et al., J. Biol. Chem. 278: 4010-4014, 2003;Wu, S. C. and Wong, S. L., J. Biol. Chem. 280: 23225-23231, 2005), because part of the biotin binding site comes from a neighboring subunit (Chilkoti, A. et al., Proc. Natl. Acad. Sci. USA 92: 1754-1758, 1995; Sano, T and Cantor, C. R. Proc. Natl. Acad. Sci. USA 92: 3180-3184, 1995).
Efforts have been made to reduce the multiple binding issues of streptavidin tetramers. Single mutations in the biotin binding site have been identified that reduce biotin binding affinity dramatically (Qureshi, M. H. et al., J Biol. Chem. 276: 46422-46428, 2001; Chilkoti, A. et al., Proc. Natl. Acad. Sci. USA 92: 1754-1758, 1995; Klumb, L. A. et al., Biochem. 37: 7657-7663, 1998), but these mutations can still leave Kd values in the nanomolar range and can disrupt tetramerization (Qureshi, M. H. et al., J Biol. Chem. 276: 46422-46428, 2001; Wu, S. C. and Wong, S. L., J. Biol. Chem. 280: 23225-23231, 2005). The double mutant N23A, S27D has one of the weakest reported affinities for biotin (Kd7.1×10−5M) (Chen, I. and Ting, A. Y. Curr. Opin. Biotechnol. 16: 35-40, 2005) and is still a tetramer, but each monomer subunit can still bind biotinylated cells and thus the potential for cross-linking remains.